Synaptobrevin vSNAREs (Syb1 and Syb2) are coexpressed at developing motor nerve terminals and spinal cords. A, B, Frozen section of embryonic diaphragm muscles (E18.5) from WT (A) and Syb1lew/lewSyb2−/− mice (B), doubly labeled with Texas red-conjugated α-bgt for postsynaptic AChRs (red, arrowhead) and specific antibodies against a SNARE protein (green): Syb1 (VAMP1), Syb2 (VAMP2), VAMP4, VAMP7, or Vti1a. Both Syb1 and Syb2 are detected at the NMJ in the WT (A) but not in Syb1lew/lewSyb2−/− mice (B). No detectable level of VAMP4, VAMP7, or Vti1a was found at the NMJ in WT or Syb1lew/lewSyb2−/− mice. C, Diaphragm muscle sections (E18.5) were triply labeled with AlexaFluor-647-conjugated α-bgt, anti-Syb1(rabbit polyclonal, green), and anti-Syb2 (mouse monoclonal, red) antibodies. Merged image shows that Syb1 and Syb2 are coexpressed at the NMJ in the WT (C, left panels) but absent in Syb1lew/lewSyb2−/− mice (C, right panels). D, Western blot analyses of spinal cord homogenates at E18.5 (WT and Syb1lew/lewSyb2−/−) and P14 (WT). Syb1 expression is markedly increased at P14, compared with that at E18.5. Scale bars: A, B, 20 μm; C, 10 μm.